Cartilage disorders are highly debilitating disorders including, for instance, articular cartilage trauma, meniscus injury, chondrogenesis disorders and arthritis. There are at present no optimal therapies available for treating these disorders. Cartilage tissue is neither innervated nor penetrated by the vascular or lymphatic systems and it is generally believed that due to this lack of a vasculature, damaged cartilage tissue does not receive sufficient or proper stimuli to elicit a repair response. Repair of arthritic joints thus requires orthopaedic surgery to replace the worn-out joints by a prosthesis or by a biological graft. Particularly arthritis is an enormous medical and economic problem.
Current approaches for cartilage repair rely on removal of tissue debris, access to the wound healing system of bone by penetrating the subchondral bone plate, and tissue transplantation and cell based therapies. Current clinical therapies typically involve autologous cells. Examples of such therapies are autologous chondrocytes implantation (ACI) and mosaicplasty (also known as autologous osteochondral grafts). Due to severe drawbacks, both therapies can currently only address a limited share of the cartilage repair market.
For mosaicplasty, a major disadvantage is the limitation to small defects due to limited availability of donor tissue for transplantation. For ACI, drawbacks include the necessity to perform two surgical operations, one for harvesting cartilage tissue, and another for implantation of in vitro expanded chondrocytes obtained from the harvested cartilage tissue. Apart from the fact that high costs are involved, the ACI process is long since in vitro cell expansion is necessary, during which cartilage cells de-differentiate, and lose their phenotype. Hence, a long rehabilitation of several months is needed following the surgical implantation procedure for the cells to regain their original phenotype. Only then true cartilage repair can commence.
Recently, a second generation ACI has been developed involving autologous chondrocytes in a biomaterial matrix. This technique solves some of the problems of ACI, particularly the long and open surgical procedure that was required in ACI. However, important drawbacks remain: two surgical procedures have to be carried out, involving high costs and long rehabilitation. One of the reasons why two surgical procedures have to be carried out is that the current processes for isolating chondrocytes from a tissue sample extracted from the patient takes a long time.
Hyaline cartilage, the most abundant form of cartilage, is glass smooth, glistening and bluish white in appearance and of this form of cartilage articular cartilage is the most common. Articular cartilage covers the ends of long bones of synovial joints. It is characterized by a particular structural organization, consisting of chondrocytes embedded in an extracellular material, typically referred to as “cartilage matrix”, which is an extracellular matrix rich in proteoglycans, collagen fibrils, other proteins, and water. Chondrocytes are the only cell type found in normal articular cartilage but contribute less then 2% of the wet weight in human healthy adult cartilaginous tissue.
The extracellular matrix of cartilage tissue consists predominantly of cartilage specific proteoglycan molecules with highly negatively charged sulphated glycosaminoglycan (GAG) side chains, as well as type II collagen fibrils. The GAG side chains are able to bind water molecules, thereby sequestering water and generating an internal swelling pressure within the cartilage matrix. These hydrogel-like properties are essential for the interstitial fluid flow patterns observed inside the matrix during functional loading of cartilage, at which point water is forced out of the tissue to an amount that allows the negatively charged GAG chains to repel each other. Upon release of the compressive load, water is imbibed back into the tissue matrix. The collagenous network, together with water bound GAG, enables articular cartilage to withstand large compressive loads which gives the tissue its unique function in synovial joints: smooth and pain-free articulation, spreading of the applied load onto the subchondral bone and absorbing mechanical shocks.
In normal cartilaginous tissue, proteoglycans are slowly but continuously turned over, the degraded molecules are released from the cartilage and are replaced by newly synthesized components. It is the coordinate control of synthesis and degradation of the matrix components by the chondrocytes that maintain normal cartilage.
After a tissue sample of the cartilage of the patient has been extracted, the chondrocytes present in that sample have to be isolated from the extracellular matrix before they can be expanded and implanted with the aim of repairing a cartilage tissue defect. Enzymatic liberation of cells located within an extracellular matrix, requires diffusion of the enzyme to the substrate (e.g. collagen), digestion of collagen, and liberation of the cells.
Known procedures for chondrocyte isolation are carried out by incubating a cartilage tissue sample with a solution of collagenase for a period of from 16 to 22 hours. The current belief is that shorter incubation times do not produce sufficient cell yields for expansion and tissue repair purposes, whereas longer exposure to collagenase is believed to compromise cell viability. The shortest incubation time described in the art appears to be 2 hours (Jakob et al., Connective Tissue Research, 44 (2003), 173-180). Digestion was never terminated before 2 hours.